immunology and the rheumatic diseases

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Eular On-line Course on Rheumatic Diseases module n4Hendrik Schulze-Koops, Alla Skapenko, Andrew Cope

IMMUNOLOGY AND THE RHEUMATIC DISEASES

Hendrik Schulze-Koops1, Alla Skapenko

1, and Andrew P. Cope

2

1

Division of Rheumatology, Medizinische Poliklinik Innenstadt, Ludwig Maximilians UniversityMunich. Pettenkoferstrae 8a, 81336 Mnchen, Germany

2The Kennedy Institute of Rheumatology, Faculty of Medicine, Imperial College London, 1 Aspenlea

Road, Hammersmith, London W6 8LH, United Kingdom

Address for correspondence: Hendrik Schulze-Koops, Division of Rheumatology, Medizinische

Poliklinik Innenstadt, Ludwig Maximilians University Munich. Pettenkoferstrae 8a, 81336 Mnchen,

Germany. Tel: +49 89 4160 3579; Fax: +49 89 5160 4199; e-mail: [email protected]

muenchen.de

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Learning objectives

After following this module, the student will be able to

identify the cellular components of an immune response

describe and explain the principal pathways leading to autoimmune inflammation

discriminate T cell subsets based on their phenotype and function

evaluate the contribution of T cells and T cell subsets to rheumatoid inflammation

know the different pathways of T cell subset differentiation

recognize pathways of immune regulation

discriminate NK cell subsets based on their phenotype and function

evaluate the contribution of NK cells and NK cell subsets to rheumatoid inflammation

discriminate B cell subsets based on their phenotype and function

evaluate the contribution of B cells, B cell subsets and autoantibodies to rheumatoid

inflammation

1. Introduction

The immune system has evolved over millions of years to defend the host against invading

pathogens. In lower organisms, this system comprises, among other elements, microbicidal peptides.

During the process of evolution into higher organisms, the immune response has evolved into a far

more complex set of cellular and soluble components that strive not only to orchestrate robust and

rapid responses to the invader, but also to imprint each attack in its memory. This provides organisms

with mechanisms with which to fend off subsequent attacks with far greater efficiency. Nevertheless,

the very nature of the molecular building blocks which make up all living organisms means that the

immune system cannot discriminate between the invading organism and host tissues. Thus, the

capacity to recognize foreign pathogens comes at a cost namely a huge propensity for reactivity

towards self a response which we call autoimmunity. To counter this destructive potential,mechanisms have evolved that keep these auto-aggressive pathways in check. These regulatory

pathways are collectively known as immunological tolerance, and are imposed upon the host immune

system through cell fate decisions during development of T and B lymphocytes of the immune system

or by the silencing of lymphocyte activation through either cell intrinsic or extrinsic pathways (Figure

1).

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Figure 1: Mechanisms of tolerance to self. These pathways involve cell fate decisions, leading to

apoptotic cell death, most commonly occurring during lymphocyte development and selection in the

thymus (for T cells) or bone marrow (for B cells), as well as cell intrinsic (anergy) and extrinsic

(regulation by other cells or soluble factors) mechanisms that lead to functional hyporesponsiveness

to antigen receptor engagement.

Ly: lymphocyte; R: regulatory cell

The autoimmune rheumatic diseases are among the commonest inflammatory immune mediated

disorders, and have become prototype disease entities for defining the molecular and pathological

basis of chronic inflammatory syndromes. As a group, they should be considered as somewhat

distinct from the traditional organ specific autoimmune disorders such as type I diabetes, celiac

disease and autoimmune thyroid disorders by virtue of their systemic nature and the dominant

inflammatory phenotype, driven by persistent cellular activation, the production of inflammatory

mediators and the proliferation and ultimate destruction of stromal tissues within multiple organs and

musculoskeletal structures. Why the rheumatic diseases target specific tissues remains a mystery.

Nonetheless, it is well established that cells of the innate and adaptive immune system are key

players in the initiation and perpetuation of chronic immuno-inflammatory responses.

A comprehensive review of the immunology of all rheumatic diseases is beyond the scope of a single

module. However, a detailed overview of the immunobiology of rheumatoid arthritis(RA)

encapsulates many of the key features of chronic inflammatory disorders encountered in the

rheumatology clinic. To this end we review the cellular and molecular components of the immune

system and their contributions to pathogenesis using RA as the prototype disease. Emphasis is

placed particularly on the biology of lymphocyte subsets and antigen presenting cells.

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2. Initiation of the immune response

Over the last decade the pathways that permit the host immune system to recognize and respond to

invading pathogens have been defined in greater depth. Principle among these are the Toll-like

receptors(TLR) that respond to pathogen derived products known as pathogen associated molecular

patterns, or PAMPs (Figure 2). It is likely that these and other ligand-receptor pathways play a major

role in initiating inflammatory cascades, through the activation of key intracellular signaling molecules,

the best known being the nuclear factor for activation of B(NF- B), the mitogen activated protein

kinases(MAPK) ERK, JNK and p38, and the interferon response factors (IRFs). These cascades lead

to the transcription of inflammatory genes such as cytokines (e.g. interleukins(IL) and growth factors),

chemoattractants, components involved in prostaglandin, leukotriene and nitric oxide biosynthesis

(e.g. cyclo-oxygenase, lipoxygenase and nitric oxide synthase) and tissue proteinases (e.g. matrix

metalloproteinases). The dependence of adjuvant arthritis and streptococcal cell wall arthritis in

rodents on these TLR-mediated pathways further underscores their importance, and suggests that

through expression of TLRs synovial joint stromal cells are peculiarly sensitive to stimulation via these

receptors. It is intriguing to speculate that at sites of tissue damage, endogenous TLR ligands, such

as heat shock proteins or fibronectin fragments, could amplify and sustain these inflammatorycascades long after the offending pathogen has been eradicated.

Figure 2: Receptors for pathogen associated molecular patterns the Toll-like receptors (TLR). The

growing family of TLRs has been documented in some depth, each receptor responding to ligands

that are quite distinct molecular entities. Whilst they are integral to innate immunity, serving to

respond to foreign pathogens, an increasing number of endogenous ligands derived from host tissues

have been described.

dsRNA: double-stranded RNA; ssRNA: single-stranded RNA; LPS: lipopolysccharide; hsp: heat shock

protein; CpG: Cytosin-phosphatidyl-Guanosin

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This is possible because of chemokines produced on the one hand by resident stromal as well as

infiltrating cells, including IL-8/CXCL8, RANTES/CCL5, MIP-1/CCL3, SDF-1/CXCL12, IP-

10/CXCL10, and MCP-1/CCL2, and the upregulation on endothelium of cell surface adhesion

molecules, including intracellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1

(VCAM-1) and E-selectin. C-X-C, C-C, C and C-X3-C chemokines all play a role, exerting chemotactic

activity towards neutrophils, lymphocytes and monocytes, but also influencing the topology of

inflammatory infiltrates. In synovial joints, macrophages are likely to be the dominant source of suchfactors. Crucially, the expression of chemokine receptors such as CCR4, CCR5, CXCR3 and

CX3CR1 on inflammatory cell subsets will contribute to the selectivity of cellular recruitment. The

regulation of the persistence of cells in inflamed joints, perhaps through the expression of survival

factors by tissue stroma, and the pathways that regulate egress of cells from inflammatory tissues are

equally important, but far less well understood. These events characterize the acute phase of an

innate immune response, a key checkpoint, which precedes the progression to subsequent events

that herald the onset of the chronic inflammatory phase.

3.3 T cells

T cells originate from the common lymphoid progenitor in the bone marrow and migrate as immature

precursor T cells via the bloodstream into their primary lymphoid organ, the thymus (Figure 3). Here,

T cells pass through a series of distinct maturation steps that include the rearrangement of their

antigen receptor (T cell receptor, TCR) genes and a distinctive set of changes in the expression of cell

surface receptors, such as the CD3 signaling complex and the co-receptors CD4 and CD8. During

maturation, more than 98% of the thymocytes die by apoptosis, as the developing T cells undergo

positive selection for their TCRs compatibility with self-major histocompatibility (MHC) molecules, and

negative selection against those T cells that express TCRs with high affinity for autoantigenic

peptides. The expression of a broad repertoire of peripheral tissue self antigens is coordinated, at

least in part, by the AIRE gene, deficiency of which causes generalized autoimmunity

polyendocrinopathy in mouse and man. T cells that survive selection lose expression of either CD4 or

CD8, increase the level of expression of the TCR, and leave the thymus to form the peripheral T cell

repertoire. Mature post-thymic T cells are therefore characterized by the expression of a disulfide-

linked heterodimeric TCR, the CD3 complex consisting of four invariant transmembrane polypeptides

(designated ), and one of the co-receptors, CD4 or CD8 .

Whereas the TCR confers antigen specificity to the T cell, the CD3 complex mediates signaling, and

is also necessary for the surface expression of the TCR. The TCR/CD3 complex is associated with a

largely intracytoplasmic homodimer of -chains that are critical for maximum signaling. The co-

receptors, CD4 and CD8, bind to invariant sites of the MHC class II or I molecules on antigen

presenting cells(APC), respectively, stabilize the MHC/peptide/TCR complex during T cell activation

and, thus, increase the sensitivity of a T cell for activation by MHC-presented antigen by

approximately 100 fold. The cytoplasmic domains of CD4 and CD8 are constitutively associated with

the src-family tyrosine kinase p56lck, which phosphorylates particular recognition motifs within the CD3

complex (denoted immunoreceptor tyrosine-based activation motifs, ITAMs), thereby promoting T cell

activation.

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Figure 3: Schematic representation of T cell development. T cells originate from the common

lymphoid progenitor cells in the bone marrow. They migrate as immature precursor T cells via the

bloodstream into the thymus, which they populate as thymocytes. The thymocytes go through a series

of maturation steps including distinct changes in the expression of cell surface receptors, such as the

CD3 signaling complex (not shown) and the coreceptors CD4 and CD8, and the rearrangement of

their antigen receptor (T cell receptor, TCR) genes. More than 98% of the thymocytes die during

maturation by apoptosis, as they undergo positive selection for their TCRs compatibility with self-major histocompatibility molecules, and negative selection against those T cells that express TCRs

reactive to autoantigenic peptides. In humans, the vast majority of peripheral blood T cells express

TCRs consisting of and chains ( T cells). A small group of peripheral T cells bears an

alternative TCR composed of and chains ( T cells). and T cells diverge early in T cell

development. Whereas T cells are responsible for the classical helper or cytotoxic T cell

responses, the function of the T cells within the immune system is largely unknown. T cells that

survive thymic selection lose expression of either CD4 or CD8, increase the level of expression of the

TCR, and leave the thymus to form the peripheral T cell repertoire (from Skapenko A. et al., (2005)

Arthritis Res Ther 7 S2:4-14, with permission).

In humans, the vast majority of peripheral blood T cells express TCRs consisting of and chains

(T cells). T cells mediate the classical helper or cytotoxic T cell responses. The TCR and

polypeptide chains each consists of a variable (V), a joining (J) and a carboxy-terminal constant

region (C). Extensive somatic DNA recombination of V and J region gene segments is responsible for

the enormous structural TCR diversity that is required for reactivity to the huge arsenal of potential

antigens, estimated to be of the order of 1015specificities. The TCR loci are organized in a way that

TCR diversity is concentrated on the third hypervariable regions (complementary determining region-

3, CDR3) of the TCR and chains. The CDR3 regions of the TCR and chains form the center

of the antigen binding site of the TCR. An TCR, however, does not bind antigen directly (in

contrast to a B cell receptor), but recognizes small peptide fragments that have been generated from

protein antigens.

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Upon proper activation, naive T cells proliferate and differentiate into specialized effector cells.

Differentiation of T cells is characterized by a number of phenotypic and functional alterations, such

as a reduction in activation requirements, alteration in migratory capacities, changes in life span, and

secretion of effector cytokines (for example, IL-4 and interferon(IFN)- ) or expression of other effector

functions. Most activated naive T cells become short lived effector cells, but some enter the long lived

memory T cell pool. Memory T cells, in humans, can be characterized by the expression of the short

isoform of CD45, CD45RO. Memory cells respond more rapidly to antigen challenge, express adiverse array of effector functions and do not require costimulation for activation. Thus, memory T

cells do not depend on the interaction with professional APC for activation, provided their specific

antigen can be presented in the context of the appropriate MHC molecules by non-professional APC.

3.3.2 Cytotoxic (CD8 ) T cells and rheumatic d iseasesCD8 T cells play a major role in immune responses. Their natural function is related to protection

against viral infections and tumors. CD8 T cells perform this function by cytotoxic damage of target

cells expressing MHC class I molecules and the relevant antigenic peptide. As almost all cells

express MHC class I molecules, it is clear that there is great potential for tissue damage. In addition,activated CD8+ T cells can produce very high levels of tumor necrosis factor(TNF) and IFN- , which

may contribute directly and/or indirectly to target cell destruction in autoimmune diseases.

Although the importance of CD8 T cells in the pathogenesis of RA has not extensively been

evaluated, some recent evidence suggests that autoreactive CD8+ T cells might contribute to

rheumatoid inflammation. Whereas the number of CD8 T cells in the peripheral blood of RA patients

is not substantially different compared to healthy controls, the population of circulating CD8+ T cells

shows a remarkable alteration in their TCR repertoire. This alteration is particularly evident in a

subgroup of CD8+ cells expressing CD57. CD8+CD57+T cells accumulate with the duration of the

disease in the peripheral blood and the synovial fluid and show especially high levels in knee joint

fluid and joint adjacent bone marrow. Although oligoclonal expansion is a common feature of the

CD8+CD57+T cell population, the extent of oligoclonality involving V 3 TCR gene segments in RA is

striking: 50% of the RA patients have evidence for oligoclonality in the V3 TCR family compared with

4% of healthy controls. In addition, unrelated RA patients were identified to carry clonally dominant

CD8 T cell receptors that were identical in their amino acid sequence, suggesting selection by a

common antigen. Predominant CD8 T cell clones from the synovial membrane could be followed in

serial samples over almost one year. As the identity of the antigen(s) has not been defined, it remains

to be shown whether these CD8 cells were selected for example by self-antigen relevant to the

pathogenesis of RA, or by an environmental antigen independent of the disease. In fact, enrichment

of CD8 T cells specific for epitopes from the EBV lytic cycle proteins was seen within the synovial fluidfrom patients with RA, and also from patients with psoriatic arthritis and osteoarthritis. CD8 T cells

specific for CMV, EBV, and influenza virus were enriched in the synovial fluid compared with

peripheral blood in RA patients. Clonal or oligoclonal populations of CD8 T cells were found to

dominate the responses to these viral epitopes in the synovial fluid from RA patients. These

observations may support the hypothesis that restricted TCR usage by large populations of virus

specific T cells provides one explanation for the presence of clonally expanded CD8 T cells within the

joints of patients with inflammatory arthritis. Therefore, T cell clonality at a site of inflammation may

reflect enrichment for memory T cells specific for foreign antigens rather than proliferation of

autoreactive T cells specific for self-antigen.

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Thus, the precise function of oligoclonally expanded CD8 T cells in RA remains to be determined. Of

interest, oligoclonally expanded CD8 T cells expressing TCRs encoded by the V12 gene were

shown to be autoreactive, since they recognized autologous, but not allogeneic antigen-presenting

cells. However, as these autoreactive CD8 T cells secreted IL-4 and IL-10, they might be involved in

regulation of immunityrather than aggravation.

Regardless of the antigen specificity of clonally expanded CD8 T cells in RA, synovial CD8 T cellshave been implicated in disease progression by two different observations: CD8 T cells from the

synovial fluid of RA patients include significant amounts of IFN-producing effectors cells that might

contribute to sustained inflammation by secreting pro-inflammatory cytokines. It has also been shown

that CD8 T cells may regulate the structural integrity and functional activity of germinal center-like

structures in ectopic lymphoid follicles within the synovial membrane. Activated CD8 T cells might

therefore be involved in aggravating pathologic responses in rheumatoid synovitis.

Although the accumulated data may indicate a role of CD8 T cells in rheumatoid inflammation, studies

in animals deficient for CD4 or CD8 have clearly demonstrated limited importance for CD8 T cells in

initiating and maintaining autoimmune inflammatory arthritis. Whereas B10.Q mice lacking CD4 areless susceptible to collagen induced arthritis(CIA), but not completely resistant, the CD8 deficiency

has no significant impact on the disease. No difference in the development of late occurring relapses

was noted. Moreover, in mice transgenic for the RA-susceptibility gene HLA-DQ8, CD4 deficient mice

were resistant to developing CIA, whereas CD8 deficient mice developed disease with increased

incidence and greater severity. These data indicate that CD8 T cells are not only incapable of

initiating CIA but may have a regulatory/protective effect on autoimmune inflammation. In line with this

interpretation are recent observations of a profound anti-inflammatory capacity of in vitroexpanded

CD8 positive, CD28 negative T cell clones from the rheumatoid synovium. Upon adoptive transfer into

NOD/SCID mice engrafted with autologous or HLA class I-matched synovial tissues from patients with

rheumatoid arthritis, these cells inhibited the production of IFN-, TNF, and chemokines.

Taken together, there is ample evidence that CD8 T cells may play a role in rheumatic diseases,

however, their precise role as effectors and/or regulators of rheumatoid inflammation remains to be

clarified.

3.3.3 Helper (CD4 )T cells and rheumatic diseasesIt has become clear that the mechanisms resulting in the destruction of tissue and the loss of organ

function during the course of an autoimmune disease are essentially the same as in protective

immunity against invasive microorganisms. Of fundamental importance in initiating, controlling and

driving these specific immune responses are CD4+T cells. CD4 + T cells are activated by an antigen,i.e. peptide, recognized specifically by their TCR if presented in the context of a specific MHC class II

molecule on the surface of an APC. Once activated, CD4+ T cells differentiate into specialized effector

cells and become the central regulators of specific immune responses. CD4+ T cells, therefore, have

been implicated in playing a central role in RA for a number of reasons (Table 1). For example,

activated CD4+ T cells can be found in the peripheral circulation of patients with clinically active

disease and, moreover, are dominant in the inflammatory infiltrates of the rheumatoid synovium

(Figures 4, 5). The most compelling evidence, however, implying a central role for CD4+ T cells in

propagating rheumatoid inflammation remains the strong association of RA with particular MHC class

II alleles, such as subtypes of HLA-DR4, that contain similar amino acid motifs in the CDR3 region of

the DR-chain.

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Although the exact meaning of this association has not been resolved, all interpretations imply that

CD4+ T cells orchestrate the local inflammation and cellular infiltration, following which a large number

of subsequent inflammatory events occur. Supportive of the notion of a major role of CD4+ T cells in

the pathogenesis of RA is the significant clinical success of an inhibitor of T cell activation in patients

with RA (CTLA4-Ig, Abatacept), as demonstrated in recent studies. Finally, the induction of tissue

damaging autoimmunity in animal models of autoimmune diseases by transfer of CD4+ T cells from

sick animals into healthy syngeneic recipients can be regarded as further evidence of the importanceof CD4+ T cells in autoimmunity.

Figure 4: Rheumatoid inflammation is characterized by an accumulation of activated CD4 T cells.

Mononuclear cells form the peripheral blood of a healthy individual and from the peripheral blood (PB)

and the synovial fluid (SF) from a patient with active rheumatoid inflammation were stained with

fluorochrome labeled mAbs to CD4 and HLA-DR as an indicator of T cell activation. Activated (HLA-

DR positive) cells within the gated population of CD4 positive T cells are indicated in blue. The

numbers indicate the frequencies of activated CD4 positive T cells.

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Figure 5: CD4 positive T cells infiltrate the rheumatoid synovium. Sections from the inflamed

synovium from a patient with active disease are illustrated after staining with a mAb to CD3 (left

panel) or CD4 (right panel). The cellular infiltrate consists of CD3 and CD4 positive helper T cells.

Original magnification: 200.

3.3.4T cells In humans, a small group of peripheral T cells bears an alternative TCR composed of and chains

(/T cells). and T cells diverge early in T cell development in the thymus. The function of the

T cells within the human immune system is largely unknown. In contrast to TCRs, TCRs

appear to recognize antigen directly, similar to immunoglobulins (Ig), but do not require presentation

by an MHC protein or other molecules and do not depend on antigen processing. In fact, some T

cells may recognize nonpeptide molecules such as lipoglycans derived from bacteria or

phosphorylated lipid derivatives of mycobacteria. The diversity of the

TCR is limited, suggesting

that the ligands for the TCR are conserved and invariant. Some T cells do not express CD8 or

CD4, while others express the CD8 chain but no CD8 . Because of their distinct antigen

recognition pattern, their preferential localization in the epithelium and their ability to secrete a variety

of cytokines and to mount robust and rapid cytolytic responses, T cells may contribute to the first

line of host defense, arguably at the intersection between innate and adaptive immunity. On the other

hand, T cells have also been shown to recognize self-peptides, such as stress-associated antigens

expressed on epithelial cells, tumor lines and primary carcinomas. Recognition of self-peptides and

the production of cytokines early during an immune response indicate that T cells might play a role

in the development of an immune response against self-tissue. In fact, recent studies have indicated

that T cells promote B cell-mediated systemic autoimmune diseases in MRL/lpr mice, a model

system for systemic lupus erythematosus. On the other hand, T cells may also play a role in

controlling immunity, as mice deficient in T cells have exaggerated responses to pathogens and,

notably, to self-tissues. Moreover, T cells can tolerize pathogenic autoimmune T cells in the

nonobese diabetic(NOD) mouse and in a rat autoimmune uveitis model. Together, the data suggest

that T cells might play a role in regulating an autoimmune response, although the precise outcome

appears to depend on the particular T cell clone.

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3.3.5T cells and rheumatic diseasesT cells have been implicated in the pathogenesis of rheumatoid arthritis(RA) by several

observations. In RA patients, circulating T cells show an activated phenotype, as indicated by

reduced expression of the CD3 complex and the Fcreceptor III (CD16), and elevated levels of MHC

class II. This phenotype, however, is not unique to RA as the majority of peripheral T cells alsohave an activated phenotype in healthy individuals. However, whereas the data with regard to the

frequencies of peripheral T cells in RA and their association with disease activity are highly

controversial, an increase in T cells in the synovial infiltrates appears to be a characteristic of

rheumatoid synovial inflammation. In one study, up to 14% of the infiltrating synovial T cells were

identified as T cells, and synovia with increased TCR cells had an increased tissue

inflammation score compared to RA synovia with few T cells. Importantly, the inflamed synovium

of RA patients contained T cells, which expressed particular V chains, for example V 3 or V1. As

the frequencies of V3 or V1 expressing T cells in the peripheral circulation of the same patients

were lower than in the synovium, these data suggest that T cells are clonally expanded in the

joints, presumably in response to their locally expressed specific antigen. Alternatively, V3 or V1

expressing T cells might have preferentially migrated into the synovium, where they could have

contributed to inflammation. Taken together, some phenotypic abnormalities might suggest a role of

T cells in the pathogenesis of RA. However, the function of T cells, in particular of synovial T

cells, and their contribution to rheumatoid inflammation is still as elusive as is the nature of their

specific antigen(s).

3.3.6 Natural Killer (NK) cell subsets

Natural killer(NK) cells are a subset of lymphocytes found in blood and lymphoid tissues which are

derived from bone marrow and appear as large granular lymphocytes because of their numerous

cytoplasmic granules. Although lacking antigen specific TCRs they can be thought of as primitive

cytotoxic lymphocytes since they possess the capacity to kill tumor cells or cells infected with virus.

This killing pathway is not restricted to conventional MHC restriction elements, but when cells are

activated utilizes CD16 (FcRIII), one of the receptors for the Fc portion of Ig-molecules. This form of

killing is called antibody-dependent cell-mediated cytotoxicity(ADCC). While comprising a key

component of the innate immune response, especially towards viral infections, NK cells are likely to

contribute to chronic inflammatory reactions through the production of cytokines such as IFN-, whose

expression is regulated by IL-12 and IL-18. Indeed in the RA synovial joint, NK cells may be the most

abundant source of this cytokine.

A fourth subset of T lymphocytes are the NKT cells. Unlike conventional NK cells, this lymphocyte

subset expresses a semi-invariant V24-J18 TCR, which recognizes glycolipid antigens presented

by DC in the context of MHC class I-like CD1d molecules. NKT cells can also be activated through

CD16. Whilst these cells are found at sites of inflammation such as the RA synovial joint, albeit at low

levels, their contribution to the inflammatory process is less clear. Nevertheless, NKT cells express

cytokines capable of directing the differentiation of CD4 T cells to either Th1 or Th2 lineage (see

below for more details), and as such could be considered as key players that link innate and adaptive

immune responses. Whilst NKT cells have been shown to play an anti-inflammatory role in some

contexts through the expression of IL-4 and IL-10, recent evidence suggests that they could also

contribute to the inflammatory process through expression of IL-17 and TNF.

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4. Pathways of T cell differentiation

4.1 Th1 and Th2 cell subsets

Great efforts have been undertaken to delineate the nature of the CD4+ T cells involved in rheumatoid

inflammation. Whereas the specific antigen(s) recognized by the autoreactive CD4 T cells is still

unknown in RA, much progress has been made in defining the phenotype and function of thosepathogenic CD4+ T cells. In 1986, it was discovered that repeated antigen-specific stimulation of

murine CD4+ T cells in vitroresults in the development of restricted and stereotyped patterns of

cytokine secretion profiles in the resultant T cell populations. Based on these distinctive cytokine

secretion pattern and concomitant effector functions, CD4+ T cells were divided into two major subsets

(Figure 6). T helper (Th) type 1 cells develop preferentially during infections with intracellular bacteria.

Upon activation, Th1 cells secrete the pro-inflammatory cytokines IL-2, IFN-and lymphotoxin- (LT,

TNF-). They activate macrophages to produce reactive oxygen intermediates and nitric oxide,

stimulate their phagocytic functions and enhance their ability for antigen presentation by up-regulation

of MHC class II molecules. Moreover, Th1 cells promote the induction of complement fixing,

opsonizing antibodies and of antibodies involved in antibody-dependent cell cytotoxicity, e.g. IgG1 inhumans and IgG2a in mice. Consequently, Th1 cells are involved in cell-mediated immunity. Immune

responses driven by Th1 cells are exemplified by the delayed type hypersensitivity (DTH) reaction.

Th2 cells predominate after infestations with gastrointestinal nematodes and helminths. They produce

the anti-inflammatory cytokines IL-4 and IL-5 and provide potent help for B cell activation and Ig class

switching to IgE and subtypes of IgG that do not fix complement, e.g. IgG2 in humans and IgG1 in the

mouse. Th2 cells mediate allergic immune responses and have been associated with down

modulation of macrophage activation, which is conferred to largely by the anti-inflammatory effects of

IL-4. Th2 cells can also secrete IL-6, IL-10 and IL-13. However, in contrast to mice, those cytokines in

humans are not confined to the Th2 subset but can also be produced by Th1 cells.

Differentiation of the appropriate T cell subset is of crucial importance to the host in mounting

protective immunity against exogenous microorganisms. However, it is apparent that immune

responses driven preferentially by activated T cell subsets are also involved in the development of

pathological immune disorders. For example, atopic diseases result from Th2 dominated responses to

environmental allergens. On the other hand, many experimental models of autoimmune diseases in

animals, such as experimental allergic encephalomyelitis, insulin dependent diabetes mellitus, or CIA

are characterized by a dominant activation of pro-inflammatory Th1 cells. Substantial evidence has

also been gathered to suggest that human autoimmune diseases, such as RA might as well be driven

by preferentially activated Th1 cells without sufficient Th2 cell development to down regulate

inflammation. These findings have fostered investigations to categorize the pathogenesis of many

rheumatic diseases with respect to the polarized T cell effector subsets. Although dichotomizing

complex diseases such as inflammatory rheumatic diseases in terms of Th1 or Th2 patterns might

have been an oversimplification, the concept allowed a better understanding of the mechanisms

involved in the pathogenesis of the diseases, and might also have helped to provide the basis for the

development of novel strategies for the treatment of rheumatoid inflammation.

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Figure 6: Current view of CD4 T cell differentiation. Upon activation with specific antigen, CD4 T cells

proliferate and differentiate into either the Th1, the Th17 or the Th2 subset. Th1 cells are best

characterized by the production of IFN-, they promote cellular immunity and are involved in the

development of autoimmune diseases; Th17 cells are potent pro-inflammatory effector cells, they

secrete in particular IL-17 and mediate many aspects of autoimmune diseases in animal modesl; Th2

cells produce, among other cytokines, IL-4, mediate humoral immunity and are involved in allergic

immune responses. Whereas the Th1/Th2 dichotomy was proposed in 1986, it was recently modifiedby the identification of Th17 cells.

4.2 A new member of the T helper effector cell family

Recently, the concept of the Th1/Th2 dichotomy has been revised after the identification of a novel

CD4 T cell subset that is characterized by the expression of IL-17 (Figure 6). IL-17 producing CD4 T

cells, designated Th17 cells, comprise a T cell subset clearly distinct from Th1 and Th2 cells. Th17

cells are characterized by the expression of IL-17 (IL-17A) and also reportedly express IL-17F, IL-6,

TNF and GM-CSF but neither IFN-nor IL-4. They require IL-23 for long-term survival and, in mice,develop from naive T cells stimulated with transforming growth factor (TGF)- and IL-6. Current data

are all consistent with the notion that Th17 cells are potent pro-inflammatory T cells. A critical role of

Th17 cells in the pathogenesis of several animal models of human autoimmune diseases, such as

experimental autoimmune encephalomyelitis and collagen induced arthritis, has been demonstrated

by several experimental approaches and it appears to become clear that Th17 cells rather than Th1

cells are the driving inflammatory force behind many autoimmune inflammatory model diseases in

mice. The role of Th17 cells in humans, however, is much less clear. Moreover, the role of Th17 cells

in rheumatic diseases has not been addressed thoroughly although preliminary evidence suggests

that the cytokine, IL-17 might also play a role in RA. In this regard, IL-17 enhances IL-6 production,

collagen destruction and collagen synthesis from RA synovial explants, causes bone resorption by RAbone explants, and increases osteoclastogenesis, and fetal cartilage destruction.

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Recently, Th17 cells have been identified as the T cell subset that provides the most efficient help for

osteoclastogenesis, thereby potentially linking T cell activation to bone destruction in inflammatory

arthritides. Whether or not Th17 cells are in fact involved in the pathogenesis of rheumatic diseases

and if so in which regard remains to be shown.

The outcome of an immune reaction such as that underlying rheumatoid inflammation is dictated by

the prevailing effector T cell population that emerges during the ongoing immune response. It iscurrently believed that with the notable exception of naturally occurring Tregs, all CD4 T cells

populations in the peripheral system emerge from phenotypically uncommitted precursor T cells from

within the peripheral system. It has become evident, that the regulation of the development of the

different CD4 T cell populations is a complex system, in which cytokines play a dominant role.

Whereas IL-2 is required for the differentiation of naive cells into either CD4+T cell subset without

imposing a functional bias, priming of naive CD4+ T cells in the presence of IL-4 induces differentiation

of Th2 effector cells. In contrast, Th1 cell development occurs in the absence of IL-4 and is greatly

enhanced by IL-12. Of interest, cytokines produced by a particular subset may have a positive effect

on the development of one subset while inhibiting the generation of another. For example, IFN-, the

signature cytokine of Th1 cells, antagonizes many of the effects of Th2 cells and blocks theirdevelopment. IL-4, the signature cytokine of Th2 cells, on the other hand, inhibits Th1 cell

development and enhances the generation of Th2 cells. In mice, activation of naive T cells in the

presence of TGF-yields regulatory T cells (Tregs) (see below), whereas activation of the same cells

in the presence of IL-6 together with TGF-results in the development of Th17 cells. Thus, the

cytokine milieu appears to control the development of inflammatory (Th1, Th17) or immunomodulatory

(Th2, Treg) T cell subsets. Importantly, the effect of IL-4 and IFN-is not restricted to the regulation of

the development of Th1 and Th2 cells but extents into the generation of both, Th17 and Treg cells.

Whereas IL-4 and IFN-both potently inhibit the development of Th17 cells, IL-4 also induces the de

novo generation of peripheral Tregs. These pathways of T cell differentiation, which have been best

characterized in the mouse, are summarized in Figure 6.

Besides cytokines, other factors have been identified that control CD4+ T cell subset polarization. The

most important of such factors include the nature and intensity of costimulatory signals, in particular

via CD28 and OX40, the intensity of TCR ligation during priming, the type of antigen presenting cells,

the MHC class II genotype, minor histocompatibility complex genes, and corticosteroids or

endogenous hormones. All together, the data clearly demonstrate that T cell differentiation is

controlled at different levels that also include a complex network of positive and negative feedback

mechanisms between the individual CD4 T cell populations themselves. As many of these factors are

altered in chronic inflammation, it is of no surprise that dysregulated T cell differentiation is a common

finding in rheumatoid inflammation.

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5. Pathways of immune regulation

In as much as chronic pro-inflammatory effector T cells are necessary to drive chronic inflammation in

autoimmune diseases, the mere presence of autoreactive T cells and even their activation in

response to recognition of cognate self-antigens are insufficient for sustained autoimmune

inflammation and, thus, the development of an autoimmune disease`. Rather, the development of

autoimmune diseases requires the breakdown of immunologic self-tolerance that usually controls selfand non-self discrimination. The primary mechanism that leads to tolerance to self-antigens is thymic

deletion of self-reactive T cells. However, since some self-reactive T cells physiologically escape this

process and autoreactive CD4+ T cells are therefore present in the peripheral circulation of healthy

individuals where they retain their capacity to initiate autoimmune inflammation, negative selection in

the thymus is not enough to prevent sustained activation of self-reactive T cells in the periphery.

Thus, regulatory mechanisms in the peripheral immune system are required to protect against both,

the generation of self-directed immune responses and the consequence thereof, the initiation of

autoimmune diseases. Overwhelming evidence suggests that one such mechanism of peripheral

tolerance involves the active suppression of T cell responses by CD4+ T cells with a regulatory

capacity, of which a major subset are the CD4+

CD25+

regulatory T cells (Tregs). CD4+

CD25+

Tregsare characterized by a low proliferative capacity upon triggering with polyclonal or allogeneic

stimulation, and by their ability to suppress CD4 and CD8 immune responses via cell-contact

dependent mechanisms. Tregs are typified by the expression of an array of surface molecules, of

which several have been implicated in contributing to the suppressive function of Tregs. For example,

CTLA4 and CD25, which are upregulated on naive and memory T cells upon activation, are

constitutively expressed on the surface of Tregs. Other surface molecules that have been described

to be characteristic of Tregs include Glucocorticoid-induced tumour-necrosis factor receptor family-

related protein (GITR), lymphocyte activation gene (LAG)-3, and integrin E7 (CD103). Recently the

reduced surface expression of the -chain of the IL-7 receptor (CD127) has been suggested to be

associated with Tregs. Despite the growing list, however, of surface markers that reportedly areselectively expressed on subsets of regulatory T cells, it should be stressed, that neither any surface

receptor alone nor a combination of different surface markers has been uniformly accepted to be

suited for a reliable identification of a homogenous population of CD4+CD25+Tregs. Of importance for

human immunology, many of the surface receptors that have been suggested to be constitutively

expressed on populations of regulatory T cells in mice are regulated in human CD4 T cells upon

activation and, thus, make a precise identification, isolation and characterization of Tregs in man even

more difficult.

The most specific protein identifying Tregs to date remains the transcription factor forkehead box p3

(Foxp3). Foxp3 was first identified as the gene responsible for the defect in scurfy mice, which dieearly in life from CD4 T cell-mediated lymphoreticular disease, and was subsequently demonstrated

to be important in murine regulatory T cell development and function. Patients with the IPEX

syndrome (immune dysregulation, polyendocrinopathy, enteropathy, and X-linkedinheritance), a

clinical syndrome presenting with autoimmune diseases similar to that developing in mice after

depletion of CD4+CD25+Tregs, have mutations in Foxp3 and lack functional Tregs. This observation

provided a first correlation between regulatory T cells and T cell mediated autoimmune diseases in

man and mice caused by a genetic defect in a defined transcription factor, which is essential for the

development of the function of Tregs.

However, notwithstanding its importance in Treg biology in mice and man, Foxp3 expression is not as

selective for Tregs in man as it appears to be in mice and recent data strongly suggest that in

humans, Foxp3 is transiently expressed in CD4+effector T cells upon antigen specific activation.

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The exact meaning of this observation is currently unclear, but recent evidence suggests that the

transient expression of Foxp3 in human effector cells may correlate with a transient phase of

hyporesponsiveness of these cells associated with some regulatory capacity, potentially resulting in a

down-modulation of the immune response, thereby preventing tissue damage form uncontrolled

immunity.

CD4+

CD25+

positive Foxp3-expressing Tregs develop as a population distinct from effector T cells inthe thymus. They are termed natural occurring Tregs and, as explained above, their major function

is to control autoimmune responses and, thus, are critically involved in maintaining peripheral

tolerance. In addition to these central Tregs, other populations of CD4+T cells have been identified

that effectively control immunity to self-antigens. These subsets are not present in the thymus but

may develop in response to cognate antigen if stimulated under appropriate conditions in the

periphery from CD4+CD25- precursors. Consequently, these Tregs are termed peripheral Tregs or

adaptive Tregs. It has been show that various immunoregulatory cytokines, such as IL-4, TGF-or

IL-10 are able to foster the generation of peripheral Tregs. Once differentiated, peripheral Tregs may

express Foxp3 and CD25. In mice, peripheral Tregs can be generated and expanded in vitrowith

different protocols and they have been successfully used in therapeutic approaches to controlautoimmune responses. To date, however, reliable protocols to generate Tregs in numbers sufficient

to allow treatment of established autoimmune disease in man, have not been defined.

In rheumatic diseases, several studies have proposed that the function of Tregs is severely impaired,

suggesting in fact that a break down of Treg-mediated peripheral tolerance may have occurred that

contributed to the development of the diseases by allowing the initial autoimmune response to evolve

into sustained inflammation. However, as recent evidence has clearly established that human effector

cells transiently upregulate Foxp3, these data should be interpreted with some caution as no reliable

way to identify a pure population of human Tregs exists. Functional studies of CD25 Foxp3

expressing T cells in patients with a disease characterized by immunological activity, such as

inflammatory rheumatic disease, are at risk to have analyzed a mixture of Tregs and variable

frequencies of contaminating, recently activated effector T cells. Future studies are clearly required to

precisely determine the role of Tregs in human rheumatic diseases, to analyze their contribution to

initiation, perpetuation and regulation of the autoimmune inflammation and also to define their

potential role as a therapeutic option to down-regulate ongoing inflammation by means of

personalized cellular therapy.

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6. Towards T cell homeostasis

Together, unequivocal data display an array of abnormalities of CD4+ T cells in rheumatoid

inflammation that provide profound insights into the pathogenesis of the disease and clearly define

inflammatory rheumatic diseases and many of their associated clinical symptoms as T cell dependent

autoimmune disorders. For example, rheumatoid inflammation is characterized by a predominance of

inflammatory effector T cells whereas T cell subsets with a potential to down modulate inflammationare impaired in number and function. Undeniable evidence has been provided for the presence of the

prototype inflammatory CD4+ T cells subset, Th1 cells, in the peripheral blood and the inflamed

synovium of patients with RA and many other rheumatic diseases. Moreover, as these cells are

activated in vivoand the reduction of their activation and/or migration to local sites of inflammation by

means of a specific inhibitor to T cell migration in experimental therapeutic settings ameliorates

clinical disease activity, the data strongly imply a pathogenic role of Th1 cells in rheumatoid

inflammation. Recent data suggest, that an even more potent inflammatory T cell subset, Th17 cells,

might play a significant role in the pathogenesis of rheumatic disease. In contrast, Tregs appear

functionally impaired in many rheumatic diseases and Th2 cells that might also be able to counteract

Th1-mediated inflammation, can rarely be detected in the peripheral blood and the synovial tissue ofpatients with rheumatoid inflammation. These observations clearly emphasize the importance of CD4+

T cell subsets in the pathogenesis of rheumatic disease and substantiate the notion of a dysregulated

CD4+ T cell balance that is characteristic of rheumatic inflammation.

7. B cells

Like T cells, B cells also derive from the common lymphoid progenitor in the bone marrow. Unlike T

cells, however, B cells undergo distinctive maturation steps already in the bone marrow, that include

the rearrangement of their B cell receptor(BCR) genes and various changes in the expression of cell

surface receptors, such as CD19, CD20 and different forms of the BCR, before they are released intothe circulation as naive B cells. The primary function of B cells is to recognize macromolecules, i.e.

antigens, through their surface receptor (BCR), which may induce further maturation of the B cells into

plasma cells and result in the production of huge amounts of a soluble form of the BCR, termed

antibody (Figure 7).

B cells (via their BCR) can recognize their target antigens directly and, in contrast to T cells, they can

recognize the native, three-dimensional structure of their targets. It is generally accepted that any

given B cell can only produce one kind of BCR and, thus, that all antibodies derived from any given B

cell or plasma cell are identical. Antibodies mediate the humoral part of the specific immune

response. They are large soluble glycoproteins that belong to the Ig-family of polypeptides. Antibodies

can bind to cell surface antigens as well as to soluble antigens. Upon binding of the target antigen by

a specific antibody, the target may be neutralized, destroyed or eliminated by different mechanisms,

such as opsonization (of infectious organisms), recruitment (of host effector cells), direct

neutralization, or removal (from the circulation).

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Figure 7: Schematic representation of B cell development. B cells originate from the common

lymphoid progenitor cells in the bone marrow. In the bone marrow, they undergo a first series of

maturation and selection steps that ensure the expression of a properly folded and functional BCR on

the surface of the B cell. The first expression of the B cell surface molecules CD19 and CD20 is

characteristic of pro and pre B cells, respectively. B cells leave the bone marrow and migrate as naive

B cells expressing surface IgD, IgM, CD19 and CD20 through the peripheral circulation. Upon

encounter with their antigen in secondary lymphoid organs, B cells undergo further maturation thatinvolves somatic hypermutation, isotype class switch and affinity maturation. These steps are critically

dependent on cognate B cell/T cell interactions. During maturation in the germinal centers, B cells are

negatively selected against the ability of BCR to bind autoantigens. As a result, B cells differentiat into

memory B cells and Ig-secreting plasmablasts that may enter the bone marrow, differentiate into

plasma cells, downregulate the expression of CD20 and reside in specialized niches for many years.

An antibody consists of two identical pairs of a large and a small polypeptide chain each, denoted

heavy and light chains, respectively. There are two alternative types of light chains, denoted and ,and five different major heavy chains (, , , , and ). Whereas the light chains are made up of two

domains, the heavy chains comprise of four Ig-domains. Within all antibodies composed of a given

light and a given heavy chain, one domain of the light chain and three domains of the heavy chain are

relatively constant in amino acid composition. Thus, they do not differ substantially from one antibody

to another and are therefore known as constant (C)-regions. The remaining domain of both, each

heavy and each light chain, contains stretches of amino acids that demonstrate enormous variability

between individual antibodies. These regions are therefore termed variable or V-regions. Thus, the

structure of an antibody is ideally suited to permit its two main functions, i.e. the recognition of a

specific target from within the enormous diversity of potential antigens and the engagement of a

relatively small number of defined effector functions of the immune system.

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Whereas the C-regions determine the ability of the molecule to engage different effector functions

(such as complement activation or Fc-receptor binding), the regions with the highest degree of

variability (hypervariable regions or CDRs) come into close proximity after the folding of the Ig

molecule into its native, tertiary structure and together form the binding site for antigen.

The different heavy chains define the family or isotypesof the antibodies (i.e. IgA, IgD, IgE, IgG and

IgM) (Table 2). The isotype of an antibody, in turn, determines its effector functions. In man, two IgA(IgA1, IgA2) and four IgG (IgG1, IgG2, IgG3 and IgG4) isoforms exist that also differ in their specific

effector functions and add to the complexity of the specific humoral immune response. For example,

human IgG1 and IgG3 can effectively activate complement, whereas IgG4 has no, and IgG2 only little

complement binding activity. These differences may play an important role in determining whether an

autoantibody that is directed against self-tissue can induce the destruction of target organs by means

of complement activation or not. The nature of the heavy chain also decides which cells might be

recruited by the antibody, as specific receptors for the different Ig families exist (denoted Fc

receptors) with a distinct distribution on the surface of different cells.

Peripheral blood naive B cells that leave the bone marrow to join the peripheral B cell pool express

surface IgM and IgD. They migrate into the secondary lymphoid organs (i.e. spleen, lymph nodes,

tonsils, Peyers patches), where they are negatively selected against the ability of their BCRs to

interact with self-antigen. As a consequence, only a minority of mature B cells survives that starts to

constantly recirculate throughout the body in search of their specific antigen. Largely depending on

the nature of the antigen, B cells, upon activation, may produce just soluble IgM or also start to

undergo a series of differentiation steps. In the vast majority of B cell responses, B cells get activated

in the lymph nodes and come into close proximity with activated T cells. This cognate interaction

between B and T cells, which takes place within specialized structures called germinal centers,

initiates three distinctive maturation processes characteristic of B cells: diversity generation by

somatic hypermutation, affinity maturationand selection of alternative heavy chain genesresulting in

isotype switchingof the produced antibody.

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Receptor editing, through successive rearrangements of immunoglobulin genes, is a more recently

described mechanism of shaping the repertoire of BCR that is especially important for precluding the

appearance of self-reactive B cells. In the absence of robust anti-self reactivity, nave B cells will

differentiate into high affinity effector cells, namely memory B cells, that can elicit a specific, high-

affinity secondary immune response and, similar to the situation in T cells, have less stringent

requirements for activation, and Ig-secreting plasma cells.

Obviously, somatic hypermutation and affinity maturation both bear the risk of the development of

autoreactive BCRs. Thus, differentiation of B cells into effector cells must be strictly regulated to

ensure sufficient specific humoral immunity whilst simultaneously avoiding the production of

autoantibodies. Critically involved in regulating B cell responses are receptorligand pairs of the TNF

receptor (TNF-R)/TNF superfamily. Whereas interactions between CD40, CD27, CD134 (OX40) and

TNF-R on B cells and their respective ligands (CD40L, CD70, CD134L, TNF) on CD4 T cells promote

B cell proliferation, differentiation and Ig secretion, ligation of CD30 and CD95 negatively regulate B

cell behaviour. Most recently, another ligand of the TNF superfamily, B cell activating factor (BAFF)

has emerged as a potent regulator of multiple functions of human B cells. BAFF (also known as

BLyS) belongs to the TNF superfamily. It is produced predominantly by myeloid cells (monocytes,macrophages, DCs, astrocytes) and binds to three different receptors (BAFF receptor,

transmembrane activator of and calcium modulator and cyclophilin ligand (CAML) interactor (TACI),

and B cell maturation antigen(BCMA)) that are expressed in different ways on B cell subsets. BAFFs

ability to enhance proliferation and Ig secretion by human B cells co-stimulated through the BCR

identified BAFF as a most important regulator of B cell function. Subsequently it became apparent

that the function of BAFF is largely related to its ability to sustain survival of B cells at different stages

of differentiation and development.

7.1 B cells and rheumatic d isease humoral immunity

The role of B cells in rheumatic diseases is manifold. Obviously, B cells produce antibodies that, ifdirected to autoantigens, may initiate tissue pathology via the antibodys ability to elicit effector

functions of the immune system. Autoantibodies to tissue antigens are a hallmark of inflammatory

rheumatic diseases (Table 3) and strongly emphasize the autoimmune nature of these diseases.

Autoantibodies have clearly established their value in the diagnosis and differential diagnosis of

rheumatic diseases. For example, RA is characterized by the presence of autoantibodies, such as

rheumatoid factor(RF) or antibodies to citrullinated proteins( antibodies to cyclic citrullinated proteins,

anti-CCP) in the serum and the synovial fluid, which distinguishes the disease from other clinically

related arthritides, such as reactive arthritis, psoriatic arthritis or polymyalgia. Anti-CCP antibodies are

discussed in more detail below. The specificity of the autoantibodies for a given rheumatic disease isdifferent and it is generally not well understood why particular antigens are targeted in particular

diseases. For example, RF is an autoantibody directed to the Fc part of Ig. Why RF is frequently

found in RA (60 80% of the patients) but rarely in psoriatic arthritis (10 20% of the patients), is

unclear. On the other hand, RF can be detected in many different diseases and is by no means

specific for RA. Moreover, the presence of RF is not even necessarily associated with any underlying

disease, and RF can be detected in up to 25% of healthy individuals. Thus, most individuals with RF

never develop any rheumatic disease and the positive predictive value of RF for the development of

RA is just 24%.

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Table 3: Autoantigens in the rheumatoid arthritis

Antigen Sensitivity (%) Specificity (%)

fillagrin

hnRNP-A2

vimentin

BiP/grp78

collagen I

eIF4G1

CCP2

fibronectin

-enolase

IgM RF

HMG1/2

p68 (heavy chain BP)

glucose-6-phosphate isomerase

calpastatin

proteoglycan/aggrecan

cartilage link protein

HCgp-39

Osteopontin

Hsp60

DNAJ

IR-3 (EBV)

p205

calreticulin

gp130-RAPS

40

32

42

68

32

50

75-80

14

46

73

25-40

64

64

45

nd

nd

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In contrast to RF, anti-CCP antibodies are found almost exclusively in RA patients making them the

most specific serological markers of RA. However, although several hypotheses exist to explain this

specificity (see below), a generally accepted reason for the appearance of anti-CCP autoantibodies

specifically in RA has not been provided.

Despite their value in differentiating rheumatic disease, which suggests a role of their antigens at

some place in the pathogenesis of the diseases, the precise contribution of most of the autoantibodiesto the development of rheumatic diseases is elusive. In fact, current evidence implies that many

autoantibodies do not play a major role in the pathogenesis of the diseases. It is undisputed that

many autoantibodies may form immune complexes that can activate phagocytes such as

macrophages or endothelial cells, thus initiating or promoting inflammation. However, whether this

does in fact occur in vivo and if so whether this has a pathological consequence in the generation of

the particular disease, has not been thoroughly addressed for many of the autoantibodies. Even

autoantibodies with the highest specificity for particular diseases, such as anti-CCP autoantibodies for

RA, have not been convincingly shown to possess any potential to mediate per seaspects of

rheumatoid inflammation.

7.2 Autoantigens

While current models of adaptive immune responses would suggest that DC carry antigens derived

from damaged or dying synovial tissue, the molecular nature of disease-associated antigens has, until

recently, remained an enigma. Many RA-associated autoantigens have been described, while other

candidate autoantigens have been validated vigorously in animal models. The best described are

collagen II, HCgp-39 and more recently glucose-6 phosphate isomerase. However, when used as

recombinant antigen, none of these have been found to elicit reproducible and/or robust T or B cell

responses in a significant proportion of patients. There are several plausible explanations for this. The

most obvious are that antigens that drive autoimmune arthritis are not the same in mouse and man, or

that detectable T cell responses occur very early and are blunted in established disease. Anotherpossibility is that the autoantigens used to test lymphocyte reactivity in vitrodo not carry the post-

translational modifications (i.e. the neo-epitopes) recognized by autoantibody or antigen receptor. A

good example is the carbohydrate moieties of collagen II epitopes that serve as key TCR contacts in

collagen II immunity, and the citrullination of key arginine residues in triple helical CII peptides that

appear to be the immunodominant autoantibody epitopes.

In 1998, van Venrooij and colleagues first reported that patients with RA carried antibodies that

recognized de-iminated peptides of fillagrin, the substrate that was found to be the antigen recognized

in rat keratinized epithelium. This substrate formed the basis of the anti-perinuclear factor (APF)

assay. Using new generation anti-CCP based assays, the presence of these antibodies, nowcollectively termed anti-citrullinated protein antigens (anti-CPA), have now been shown by many

groups to be both sensitive (up to 80%) and highly specific (> 95%) for the diagnosis of RA. Indeed,

serum anti-CCP levels are stable with disease, they have been detected as early as 14 years prior to

disease onset and have been shown to be predictors of radiographic progression. Citrullination is not

specific for RA (Figure 8). Indeed, citrullination may be inflammation specific, since it has been

documented in inflamed synovium derived from patients with reactive arthritis and psoriatic arthritis,

as well as RA, but not OA. What appears specific for RA is the immune response to citrulline. A link

between anti-CCP and HLA-DRB1 alleles, specifically shared-epitope(SE) alleles, has now been

established.

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Figure 8: Citrullination of tissue antigens. The generation of modified self-proteins, commonly through

post-translational modification, provides a particularly attractive means whereby the immune system

may incorrectly recognize self proteins as foreign, leading to autoimmunity through the generation of

neo-epitope specific immune responses, including autoantibodies. Citrullination arises through post-

translational modification of arginine residues to the non-naturally occurring amino acid citrulline by a

process known as de-imination, catalyzed by a group of enzymes called peptidyl-arginyl-deiminases

(PADs). Citrullination is a common manifestation of inflammatory responses, especially thoseassociated with significant cell death. What appears to be specific to RA is the host immune response

to citrullinated protein antigens (anti-CCP), a response that may be linked to RA associated MHC

class II molecules (collectively known as the shared epitope positive HLA-DRB1 alleles).

Linkage analysis across chromosome 6 has documented a large peak with LOD scores of > 10 for

anti-CCP positive patients but not for those that do not carry these antibodies. This relationship is

independent of RF status since the SE allele frequencies in anti-CCP positive patients are twice those

of anti-CCP negative patients, even those patients who are RF positive. Indeed, the risk of carrying

SE in RF positive anti-CCP negative patients is no different to the healthy control population. These

data would be consistent with a model where T cells from patients with RA can recognize peptide

autoantigens modified by citrullination if they carry SE positive DRB1 alleles. Studies in HLA class II

transgenic mice suggest that the conversion of positively charged arginine at key residues in antigenic

peptides from candidate autoantigens to neutral citrulline is permissive for peptide binding and the

induction of antigen specific immune responses in vivo.

Finally, citrullination is widespread in multiple tissues in response to appropriate provocations.

Although the molecular basis for these triggers is poorly understood, recent data points to a link

between smoking, an environmental exposure known to be linked with RA, citrullination and

individuals carrying SE. Thus, cells derived from bronchoalveolar lavage from smokers, but not from

non-smokers express citrulline.

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The association between the development of RA and smoking has now been linked to anti-CCP

positive patients whose relative risk increases to 20 if they smoke and carry two copies of the SE

positive DRB1 alleles, as compared to patients with anti-CCP negative disease where there appears

to show much weaker or no such relationship. These data are the first to show a direct link between

environmental exposure and disease specific immune responses governed by immune response

genes. They are clinically significant because at the outset of disease they provide useful predictors of

patients who are more likely to progress to develop severe erosive joint disease.

7.3 B cells and rheumatic d isease cellular immuni ty

In addition to producing antibodies B cells also contribute cellular aspects of specific immunity. For

example, B cells may efficiently present antigen via MHC class II to CD4 T cells and thereby promote

T cell activation and control T cell differentiation. Moreover, B cells are able to regulate the

differentiation of follicular dendritic cells. As B cells can produce significant amounts of IL-6 and TNF,

B cells may manifest a clear pro-inflammatory phenotype. The recent clinical observations of a

profound amelioration of disease activity in several inflammatory rheumatic diseases after B cell

depletion with a monoclonal antibody to CD20 (rituximab) may relate to these functions of B cells

rather than to the ability of the B cells to secrete antibodies. This hypothesis is substantiated by the

fact that plasma cells, the main antibody secreting B cell subset, do not express CD20 and are

therefore not targeted by rituximab. Consequently, whilst rituximab potently destroys peripheral blood

B cells (by complement dependent cytotoxicity and, presumably, by antibody dependent cellular

cytotoxicity), serum Ig levels are largely unaffected by rituximab therapy and most autoantibody

serum titers remain relatively stable over time. In this context, it is tempting to speculate that those

autoantibodies which are reduced in the serum following anti-CD20 monoclonal antibody therapy, are

produced by CD20 expressing plasma blasts rather than by plasma cells (that are CD20 negative)

(Figure 7).

7.4 CD5+(B1) cells

A small subset of B cells does not conform to the developmental pathway sketched above. These

cells arise early in ontogeny, comprise 5 10% of the circulating B cells, have a limited BCR

repertoire due to their lack of the enzyme terminal deoxynucleotidyl transferaseand are potentially

self-reactive. They are characterized by the surface expression of IgM with little or no IgD and the

expression of CD5. These B cells are hence called CD5 B cells (or B1 cells; in contrast to the vast

majority of conventional CD5 negative B cells that are termed B2). CD5 serves to mitigate activating

signals from the BCR, thereby potentially preventing B1 B cell responses to self tissue proteins. CD5

B cells are the producers of natural IgM antibodies present in the serum, they originate form the fetal

liver, are found preferentially in the peritoneal and the pleural cavity, are self renewing and arguablyplay an important role in the first line defense of the organism. Typically, CD5 B cells respond to T cell

independent antigens, such as microbial polysaccharides, lipids or LPS. These antigens may activate

nave B cells but fail to simultaneously activate T cells. As a consequence, T cell independent

antigens do not generate a memory B cell response and also fail to induce isotype switching, both of

which are critically dependent on T cell-mediated signals.

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7.5 CD5+(B1) cells and rheumatic diseases

A role of CD5 B cells in rheumatic disease has been suggested by the observation of a coordinated

expansion of circulating CD5 B cell and T cells in younger patients with RA as well as in patients

with primary Sjgrens syndrome. However, the functional implication of such an alteration remains

hypothetical. Subsequent studies did not confirm this finding and also failed to correlate CD5 B cell

levels with several indicators of disease activity or rheumatoid factor levels. Thus, the data suggest arather trivial role of CD5 B cells in rheumatoid inflammation. Limited data supporting this conclusion

derive from a treatment trial of patients with active RA with a monoclonal antibody to CD5 coupled to

the ricin A chain (CD5-IC). Although the primary target of this approach were T cells (that express

CD5), CD5 B cells were also bound by CD5-IC and killed by the ricin A chain. Treatment with CD5-IC

resulted in transient reduction of the number of circulating T and B1 cells, however, was not

associated with a significantly increased clinical effect as compared to the placebo control. Together,

the data support the notion that CD5-IC was ineffective for treating RA and also that CD5 B cells

might have a minor if any role in the pathogenesis of rheumatic diseases.

8. Concluding remarks

There remain many unanswered questions regarding the immunobiology of rheumatic disease. For

example, what is the contribution to chronic immune and inflammatory responses of allelic variants in

immune response genes besides the MHC? Why do these aberrant responses target synovial joints

or other musculoskeletal structures, as opposed to other tissues? How can we best apply the current

knowledge to introduce into the clinical setting immunological biomarkers to facilitate diagnosis and to

monitor the impact of our therapeutic interventions? How can we use this information to define the

pre-arthritic phase of disease in the healthy population so that we can consider strategies for disease

prevention? Our understanding of the molecular basis of immune-mediated diseases has advanced

considerably over the last two decades. However, much work needs to be done to translate this

knowledge to the benefit of patients in the clinic.

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Summary

Autoimmune inflammation results from a specific immune response (e.g. T- and B-cell driven)

to self-antigens.

The main cellular players of an autoimmune response are dendritic cells, T cells and B cells.

Dendritic cells, T cells and B cells are all comprised of functionally clearly distinct subsets that

can be separated based upon the expression of different surface molecules.

Leucocyte trafficking to sites of inflammation is directed by chemokines and adhesion

molecules.

Rheumatoid inflammation is largely driven by activated CD4 T cells that express pro-

inflammatory cytokines (e.g. IFN-, IL-17).

Specific CD4 T cells (regulatory T cells) are able to down-modulate and prevent

(autoimmune) inflammation and, thus, contribute to peripheral tolerance. Evidence suggests

that their number and/or function in rheumatoid inflammation may be reduced/impaired.

B cells contribute to inflammation in rheumatic diseases by secreting pro-inflammatory

cytokines and autoantibodies and by their ability to present antigen to T cells

The presence of autoantibodies in rheumatic diseases emphasizes the autoimmune nature of

these diseases. The autoantibodies, however, may not be specific for a particular disease and

may not be involved in the pathogenesis of the diseases themselves. Their value, therefore, in

identifying the target autoantigen(s) in rheumatic disease and their potential value as targets

for biologic treatment regimens is limited.

Citrullination is a common posttranscriptional modification in inflammatory disease. The

development of antibodies to citrullinated peptides, however, appears to be characteristic and

specific for rheumatoid arthritis. The contribution of antibodies to citrullinated peptides to the

pathogenesis of RA remains elusive.

282007-2008 EULAR


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